This past year (2010-2011) our laboratory built upon the foundation begun in 2008 to explore the interface between the malaria parasite and the host immune system. We have continued our collaborative 5-year longitudinal study of 1400 children in Mali and in 2009 we identified a sub-cohort of these children, selecting those with the sickle cell trait (HbAS) and pairing them with age-matched HbAA controls. These children have been followed from 2009 to 2011, with blood samples being obtained before and after the transmission season as well as during bouts of clinical malaria;these samples provide a unique and valuable resource for studies on the development of humoral and cellular responses to blood-stage antigens of malaria parasites.From the first year of our study it was apparent that children with HbAS are significantly protected against mild malaria in this population. Because long-standing data has shown that antibodies can play a significant role in protection against erythrocytic stages of infection, we have pursued a detailed characterization of the antibody populations elicited by malaria infection in the Malian children. Initially we tested the hypothesis that the HbAS children develop accelerated antibody responses to blood-stage malaria antigens as a basis for their protection. However, we have shown that in fact their humoral responses to a number of malaria merozoite antigens are lower than their matched controls. This cannot be responsible for their increased resistance. We believe instead that the lower responses in the HbAS children may result from their lower incidence of infection. We have also shown that there is an age-dependent increase in the levels of antibodies in both HbAA and HbAS children which may contribute to their progressive development of resistance to infection. In addition to the quantitation of humoral responses to merozoite antigens by ELISA, we have purified IgG from the same children before and after the rainy season and have completed testing the ability of these preparations to inhibit parasite growth (GIA) in vitro as a functional measure of the antibody responses. In general, the GIA analysis of IgGs from the HbAS children correlates with results from the ELISA studies. During the past year we have performed extensive statistical analysis on all these results and are currently preparing them for publication. In addition, we have developed a flow cytometry assay to profile the acquisition of antibodies in these children to antigens present on the surface of parasitized erythrocytes. We are quantifying changes in these antibody populations with age and comparing their activity in HbAS and HbAA children. Concurrently we are addressing the degree of cross-reactivity of these antibodies to different laboratory lines of parasites as well as from field isolates. We predict that increased age will correlate with broader strain recognition of parasitized erythrocytes. As another aspect of our approaches to studies of proteins on the surface of parasitized erythrocytes, we have continued our collaboration with Dr. Kavita Singh on the best-characterized surface antigen of infected red cells, viz., VAR2CSA. This protein is a member of the large and complex PfEMP1 family and has been implicated in pregnancy associated malaria through binding to chondroitin sulfate A (CSA) in the placenta. In collaboration with Dr. Kavita Singh (RTB), we have produced monoclonal and polyclonal antibodies to the DBL3X domain and characterized their binding and functionality with respect to inhibition of binding to CSA in vitro. During the past year we have extended these studies to other domains of VAR2CSA and have produced a series of polyclonal antibodies in mice immunized with these recombinant proteins. These sera are currently being characterized for binding and functional activity. Recently we have shown that antibodies from Malian women but generally not men recognize some of these domains by ELISA. Antibodies from these women, but not men, also opsonize FCR3 parasites selected on CSA and presumably expressing VAR2CSA, as would be expected for an antigen associated with parasites present during pregnancy. Our second major area of investigation relates to the identification of malaria parasite-encoded antigens which could be the targets of new vaccines or drugs. We have hypothesized that there are conserved epitopes present on the infected red cell which could represent such novel targets. As one approach to addressing this problem, we have selected a new strategy using DNA aptamers to identify conserved antigenic determinants on the surface of malaria infected erythrocytes. While most blood-stage vaccine candidates have been directed to antigens of merozoites, we believe that antigens present on the surface of the infected red cell have significant advantages as potential vaccine candidates because of their exposure to the serum for long periods. However, the antigenic complexity and diversity of the surface molecules (e.g., PfEMP1) identified to date have proven daunting in terms of vaccine development. We had previously prepared several complex DNA aptamer libraries and conducted a series of repetitive selections on various targets. To analyze the selected aptamer populations we employed next-generation sequencing technologies, primarily Illumina Solexa, to obtain deep sequencing of the complex aptamers. Using this technology we have identified over 100 aptamers with specificity for infected red cells and we have confirmed this by direct binding analysis of many of the aptamers on uninfected and infected red cells;some of these aptamers actually inhibit parasite growth in vitro as well although the mechanisms involved are not known. In addition to measuring affinity of the aptamers to the red cell, we have also recently characterized the reactivity profiles of selected aptamers and shown that some recognize a variety of parasite isolates. This is an exciting new approach to elucidating relevant epitopes on the infected red cell. Once the aptamer characterization is completed, we will seek to identify the molecular targets of the selected aptamers. We have also pursued a proteomics strategy to identify novel targets of parasite growth inhibition since previous studies in Malian sera showed that antibodies to prominent merozoite antigens such as AMA1 did not make a major contribution to the GIA activity of these sera. Since it is likely that this reflects antibodies to other merozoite antigens, we have compared reactivity profiles of IgGs with high and low GIA activity and elucidated the differences using two-dimensional gels and mass spectrometry. Analysis of these results is in progress. Another area which we have continued to expand this year is transmission blocking immunity. In collaboration with PATH/MVI, we are performing mosquito membrane feeding assays (MFA). We are currently assessing whether this difficult assay can be qualified rather than validated in order to facilitate its use in preclinical and clinical transmission blocking studies. A further new effort has been the construction and testing of a new set of mouse monoclonal antibodies to the full-length P. falciparum circumsporozoite protein prepared by Dr. Sanjay Singh in Pune, India. These monoclonals have been tested by a variety of techniques and 7 have been selected for expansion. The expanded antibodies will be tested for passive protection in mice with transgenic P. berghei parasites (with Dr. Fidel Zavala and for analysis of the fine specificity of the human immune response to the RTS,S vaccine (with Dr. Chris Ockenhouse of WRAIR). FUsing our standardized blood-stage GIA, we have also collaborated with others in analysis of several different human trials of various blood-stage vaccine candidates as well as preclinical studies.